Polypeptide growth factors play an important role in stimulating proliferation of target cells which are involved in both normal cellular processes and disease states. Some examples of processes that involve cell prolifration are, for instance, epidermal tissue replacement and immune system responses. Hemostasis, which is prevention of blood loss, is a continual process requiring that vascular tissue be constantly replaced. Growth of vascular endothelial cells has a central role in a variety of physiological and pathologicalprocesses, such as angiogenesis, woundhealing, atherosclerosis, and tumor growth. In the case of vascular trauma, a complex series of events comes into operation to maintain hemostasis. Initial response to trauma include activation of platelet plugging, blood coagulation, and eventually regrowth or repair of the damaged tissue. This final step must be activated at the proper time, place and in the proper tissue. One role for polypeptide growth factors is to mediate this response in both a temporal and tissue specific manner.
Peptide and polypeptide growth factors have been isolated from many sources. These include epidermal tissue, neutral tissue, platelets, placental tissue, and others. These peptide and protein factors are distinguished by a variety of properties including target cell specificity, heparin affinity or interaction, secretory properties, and physiochemical properties such as molecular weight, charge, heat stability, pH sensitivity, and susceptibility to reducing agents.
Endothelial cell growth is stimulated by a class of polypeptide growth factors known as fibroblast growth factors (FGFs). These mitogenic factors are characterized by their heparin-binding properties. Heparin is a powerful anticoagulant agent normally found in minute amounts in the circulatory system. The FGFs fall into two distinct protein classes, acidic and basic, and have been identified in neural and other tissues Baird et al., Recent Prog. Horm. Res. 42: 143-205 (1986); Lobb et al., Anal. Biochem. 154: 1-14 (1986); Thomas et al., Trends Biochem. Sci. 11: 81-84 (1986).
Recently, endothelial cell proliferation was shown to be stimulated by a novel polypeptide growth factor that was distinct from other known polypeptide growth factors derived from fresh human platelets Miyazono et al., Biochem. Biophys. Res. Commun. 126: 83-88 (1985); Miyazono et al., Exp. Cell Res. 159: 487-494 (1985). This factor was originally called vascular endothelial cell proliferation factor and was later renamed platelet-derived endothelial cell growth factor PD-ECGF.
Platelets are a rich source of growth factors for a variety of hemopoietic and other tissues. Platelet-derived growth factors that have been identified and characterized include platelet-derived growth factor (PDGF) which stimulates fibroblast and vascular smooth muscle cell growth but has no effect on endothelial cells; transforming growth factor-.alpha. (TGF-.alpha.) which is closely related to epidermal growth factor (EGF) and can stimulate growth of epidermal cells but not endothelial cells; transforming growth factor-.beta. (TGF-.beta.) which synergistically stimulates fibroblast growth in the presence of EGF and is furthermore a potent inhibitor of endothelial cell growth; a hepatocyte growth factor; and platelet-derived endothelial cell growth factor (PD-ECGF), the subject of this invention.
A growth-promoting activity was partially purified and characterized by Miyazono et al. J. Biol. Chem. 262(9): 4098-4103. The following characteristics were used to distinguish this activity from that of previously identified growth factors: Cultured porcine vascular endothelial cells were stimulated to incorporate .sup.3 H thymidine into DNA in a dose-dependent manner upon treatment with a soluble lysate of fresh human platelets. The platelet lysate also promoted cellular proliferation by about 100% above a control culture treated with 1% fetal bovine serum. Fractionation of the lysate revealed that the activity appeared in the Mr 20,000 range on a Sephadex G-75 gel filtration column, stimulated .sup.3 H thymidine incorporation into DNA of porcine vascular endothelial cells but not NRK fibroblasts, was probably distinct from PDGF which ran at Mr 30,000 on this column and stimulated fibroblasts but not endothelial cells, was more potent when prepared from fresh platelets rather than from outdated platelets, was heat-and acid-labile, resistant to the reducing agent dithiothreitol and was sensitive to trypsin, guanidinium-HCl and urea, the latter two being denaturants. Based on these characteristics it was concluded that the growth-promoting activity was a polypeptide growth factor distinct from any that had previously been identified in human platelets.
Angiogenesis is the formation of new capillary blood vessels by sprouting from existing vessels. It is an important process in wound healing and tumor growth. Certain polypeptide factors have been identified which stimulate this process by virtue of their mitogenic or chemotactic activity for andothelial cells.
Mitogens are substances that stimulate proliferation of cells and are usually small molecules such as phorbal esters, polysaccharides, peptides, proteins or combinations thereof. Taxis is the directed migration of cells in response to an environmental cue. Chemotaxis is thus a response to chemicals in the environment. For eukaryotic cells, known chemotactic factors include histamine, amino acids, peptides, and proteins,
Polypeptide factors that elicit angiogenic responses include FGFs, TGF-.alpha., TGF-.beta., tumor neurosis factor and angiogenin (Folkman et al., Science 235: 442-447 (1987); Frater-Schroeder et al., Proc. Natl. Acad. Sci. USA 84: 5227-5281 (1987); Leibovich et al., Nature 329: 630-632 (1987)). These factors exhibit multiple effects on a wide variety of cell types. Unlike these factors, human platelet-derived endothelial cell growth factor specifically stimulates endothelial cells.
Endothelial cell proliferation inside larger blood vessels is important for the regeneration of damaged endothelium and probably plays a role in prevention of atherosclerosis. Atherosclerosis is a condition in which lipid-rich lesions or plaques develop on blood vessels and can lead to numerous vascuous diseases. Alteration in the endothelial cell layer is thought to be an early event in atherosclerosis. Platelets adhere to damaged endothelium cells and release mitogens to stimulate both endothelial and smooth muscle cell regeneration. PD-ECGF is among the mitogens that are released.
In disease associated with a reduction in platelet number, for example thrombocytopenia, PD-ECGF and other platelet mitogens and chemotactic factors may provide therapeutic benefit. Certain drugs can also induce platelet destruction or suppression. Thus, supplementing drug therapy with platelet mitogens may prevent untoward side effects.
The present source of PD-ECGF is fresh human platelets making purification of large amounts of this protein expensive and potentially risky given that human blood may contain infectious agents. Since PD-ECGF is heat labile, prior sterilization or pasteurization of blood will destroy its activity. To obtain quantities of PD-ECGF for further study and to provide therapeutic amounts, a more readily available source is desired. Cloning the PD-ECGF gene would allow the construction of expression vectors that produce large amounts of PD-ECGF.
The techniques of recombinant DNA technology are well established. While numerous manuals are available that outline the strategy and methodology for cloning genes, much of the work remains unpredictable; there is still no guarantee of success. Each cloning experiment can present its own special problems and pitfalls. A typical strategy for cloning eukaryotic genes is to isolate mRNA, to transcribe it into cDNA which is then inserted into a replicable vector, to transform a suitable host with the construct and to identify the desired clone by any number of means including functional activity or complementation, and screening with antibodies or oligonucleotides. A problem peculiar to some eukaryotic genes is finding a tissue in a state of development or differentiation that is expressing the desired gene product, that is, containing the mRNA from the desired gene. The source of PD-ECGF is platelets which are enucleated cells and thus do not contain DNA or mRNA. Hence, to clone the gene for PD-ECGF a tissue or cultured cell line that produces PD-ECGF was needed to be found.